The quantification (quantitation) of specific nucleic acids in mixtures of nucleic acids is of importance in a number of applications in molecular biology, such as gene expression analysis or during purification of specific nucleic acids from a mixture of nucleic acids. In quantification methods, the concentrations and/or the relative or absolute amounts of specific nucleic acids in samples are determined. In particular, for the analysis of gene expression, for example for measuring mRNA levels in biological samples, a reproducible and comparative method is desired. For example, it is not always possible to obtain biological samples with comparable volume, amount of nucleic acid, cellular material or the like. Different samples can, for example, comprise RNA or DNA derived from different tissues, from different organisms or individuals, or cell culture samples that have been treated with different compounds.
In addition, sensitivity and selectivity of detection and quantification of nucleic acids in biological samples is of importance. For better comparison of the quantities of specific nucleic acids in two or more different (biological) samples or the comparison of the quantities of two or more different specific nucleic acids in a sample, a normalization of the quantities of the specific nucleic acids to the input nucleic acids or a specific class of input nucleic acid has to be performed. Quantities of specific nucleic acids can, e.g., be normalized by relating these quantities to an internal standard of the sample or to the overall, i.e. total, amount of nucleic acid or to the amount of a specific class of nucleic acid in the sample.
For conventional quantification of nucleic acids in (biological) samples, quantitative (real-time) PCR (qPCR) is widely used. For RNA, particularly mRNA, quantitative real-time reverse transcription PCR (RT-qPCR) is used in this field. Different approaches for the normalization of data obtained from quantitative PCR methods have been employed. Among them is the normalization of the quantities of specific mRNAs to the quantities of one or more mRNAs of different reference genes, e.g. housekeeping or maintenance genes, such as beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine-guanine phosphoribosyl transferase (HPRT), or 28S or 18S ribosomal RNA. However, the expression levels of such normalizer genes have been shown to vary depending on experimental conditions, preparation and source (e.g. tissue or cell type) of the samples and therefore they are not reliably indicative for the input nucleic acids. It is therefore commonly required to test a range of different housekeeping genes in a laborious and error-prone procedure in order to identify those which do not change between samples under investigation.
Other approaches, for example, rely on the normalization to the total content of DNA and/or RNA or the total content of e.g. ribosomal RNA (rRNA). As the content of ribosomal RNA in biological cells and samples is also subject to variations depending on a variety of factors, normalization to rRNA is also less preferred. Methods relying on the normalization to e.g. total nucleic acid content, total RNA content or total content of genomic DNA are also limited, e.g. by variations in these contents or the quality of the nucleic acid samples. Normalization to alien or artificial molecules, e.g. in vitro transcripts, that have been incorporated into a sample (e.g. a cell extract or a sample derived from a tissue) is also not in all cases an adequate procedure, since they do not represent the nucleic acid (e.g. genomic DNA, RNA, mRNA) content in a cell.
Besides, for comparison of normalized data and reproducibility of the experimental procedures, thorough documentation of the applied experimental conditions is required. This is particularly relevant, when the quantities of the nucleic acid of interest and the normalizer nucleic acid are determined separately or using different methods.
Therefore, the technical problem underlying the present invention was to develop and to provide an improved, in particular a less laborious and error-prone, method for the normalization of quantities of nucleic acids.